Usermanual for Nikon Ti2 Eclipse

Table of content

How to turn on the system page 2

How to turn off the system page 3

Starting the software page 4

Viewing through the microscope page 5

Which camera should I use? Page 6

Live cell imaging setup Page 7

Objectives available on the system Page 8-9

Phase contrast acquisition Page 10

Köhler adjustment and phase contrast Page 11

Automated ND acquisitions Page 12

Multichannel acquisition Page 13-14

Fluorescent filter setup Page 15

Large image acquisition Page 16

Timelapse acquisition Page 17

Multipoint acquisition Page 18

Z-acquisition Page 19

Adding a scale bar Page 20

Where should I save my data? Page 21

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 How to turn on the system

1. Turn on the microscope ctrl box (1).

2. Turn on the TIS camera for fluorescence imaging (2) if needed.
3. Turn on the computer and log in using “user” and password “ ”.
4. Turn on the Fi3 color camera (4) if needed.

For live cell imaging, open the CO2 valve, turn on the OKO lab mixer and start
the heating (green boxes). See page 7 for more information.

For FRAP imaging, remember to turn on the FRAP laser and the FRAP ctrl box
(blue).

For TIRF, remember to turn on the TIRF lasers (yellow).

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4

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 How to turn off the system

1. Close the software and start a computer shutdown.

2. Turn off the TIS camera (2) and the Fi3 camera (4) (if they were
on).
3. Turn off the microscope control box (1).
4. Turn off the CoolLED (5).
5. Remember to sign the logbook and report
any issues to MIC personnel.

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4
5

3
off

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 Starting the software?

• Start “NIS-ELEMENTS
• Login using the “user” account (no
password needed).
• Select the camera you would like to use. If
you do not want to acquire any images,
select “passive mode”. For more details
about the camera options, see page 6.
• The objectives are the eyes of the system,
so make sure you use them correctly. The
60x and 100x must be used with
immersion oil. The other objectives are
DRY! The two objectives in position 5 and
6 have large working distance. These can
be used for cell culture plates and give
nice phase contrast images.

Oil objectives Objectives

Immersion oil with small with large

working distance working
and high NA. distance.

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 Viewing through the microscope

Fluorescence filter turret.

Use the multiband filter.
Press the button to
open/close the shutter.

Halogen light

Bertrans lens

To change Z speed,
press here

You can leave the 2nd

and 3rd channel on to
see both green and red!

To change the XY
CoolLed ctrl panel
5 speed, press here
 Which camera should I use?

TiS camera (2048 x 2048 pixels) Fi3 camera (2880 x 2048 pixels)

Use this camera for imaging of fluorescent Use this camera for imaging of brightfield,
samples. phase contrast and histological samples.

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 Live cell imaging setup

• Turn the small switch to open for the CO2 1

gas (1). Do Not touch any of the other
valves!
• Turn on the air pump (2). Do not touch
any of the valves on the box underneath!
• Turn on the small OKO control box (3) to
start the heating of the insert.
• Make sure you connect the correct
heating cable (4) for the objective you will
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use (60x or 100x). The red heating
element will start to warm up the
objective. For good stability allow 0,5-1 h
warm up time.
• Make sure there is enough distilled water
in the humidifier (5).
• Use the live cell insert and heating lid (6).

No no!

ON/OFF

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5

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 Objectives available on the system

Magnification Correction NA WD Contrast method

(mm) and application
10 x Air Plan Fluor 0,30 15,2 Ph 1 / phase
contrast
20 x Air Plan Fluor (coverslip correction) 0,45 7,4 Ph 1 / phase
contrast
20 x Air Plan Apochromat lambda 1,20 1,0 fluorescence

40 x Air Plan Apochromat lambda 0,95 0,21 fluorescence

(coverslip correction)
60 x Oil CFI Apo TIRF (coverslip and 1,49 0,12 TIRF / fluorescence
temperature correction)
100 x Oil CFI Plan Apochromat lambda 1,45 0,13 fluorescence

Some objectives have a correction collar (A)

to adjust for “cover slip” thickness between 0
and 2 mm (B). In order to adjust:
• Look through the eyepiece at all times.
• Turn the correction collar slightly and
refocus. Does it look sharper?
• Turn the correction collar again slightly and
refocus.
• Repeat until you get a sharp, crisp image.

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 Objectives available on the system continues…

Immersion
oil only for
60x and Objectives suitable for Objectives suitable for
100x coverslip (170 µm thick) coverslip as well as plastic
bottom dish (cell culture,
spheroids, larger samples)

Numerical
aperture
NA
1,20

0,95

1,49

1,45

0,45

0,30

Remember to adjust the

correction collar to match
the thickness of your bottom
dish or coverslip.
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 Phase contrast acquisition

• Adjust Köhler and phase contrast (see page 11) for the objective you want
to use (10x, 20x). These two objectives are optimal for phase contrast. We
also have a 40x objective (0,60 NA, Ph2) but this will only be placed on the
system upon demand.

• Select the Fi3 camera at startup.

• Press “+” on the keyboard to get the live image or press the “Freeze” icon.
Adjust exposure time and DIA lamp power to get the best result. You can
also use the AE (auto exposure) or leave the continuous autofocus on.

• Move outside your sample to adjust for the white balance (auto White).

• Acquire the images by pressing “capture” at the top.

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 Köhler adjustment and fase contrast

Always adjust Köhler after changing objective

or replacing sample.

• Close the field stop diaphragm (A) in order

to see the edges of the diaphragm.
A
• Move the condenser up and down (B) until
you see a sharp image of the diaphragm.
B
• Center the diaphragm by using the allen
key (C). C
• Open the field stop diaphragm (A) until the C
edges lie just beyond the field of view.
• Increase depth/contrast of field by
adjusting the aperture stop (arrow).

C
B

The system is equipped with a setup

for phase contrast with 10x, 20x and
40x (on request). Select the correct
phase matching the objectives
inscription. Insert the Bertrans-lens Phase contrast lens
(Bl) and use the eyepieces to align
the two rings.

Bl

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 Automated ND acquisitions

Using the ND acquisition, you can set up experiments that include:

Timelapse, Multipoint, z-series, Multichannel, Large image

• Open the ND acquisition window (icon on the top menu).

• Check the module(s) you need (A) and define each as described on the
following pages. Define the order of the experiment (B). Define also the save
path (C), preferably Biomic.
• If you need to define autofocus, open “advanced” (D).
• When you are ready, start the experiment by clicking on “Run now”.
• It is recommended to heat up the system 0,5-1 hour before starting timelapse.

D 12
 Multichannel acquisition

• Check the fluorescence on your sample using the oculars. Check each
fluorescent channel in live view and set the exposure time and coolLED
intensity for each channel. Changes with exposure time will automatically
be saved. If you see the red exclamation mark “!” behind the name of the
channel, it means that you have change the LED setting. To save this new
intensity, right click on the “!” and “assign current microscope setting”.
• To set up an automatic acquisition, see next page.

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 Multichannel acquisition continues…

• Open the ND acquisition window (icon on the top menu).

• Check the lambda window and tick the optical configurations
you need.

• Use the “M” in the left toolbar to start capturing with the
option to focus manually. Click “continue” to record the image.
• Use the “A” to automatically capture all the channels. You
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might want to define “autofocus” in the ND acquisition.
 Fluorescence filter setup

The system comes with two filter turrets and by

combining them we get the following setup:

Multi exitation filter for coolLED is used for

the following filters: dapi, GFP and mCherry.

Filter name Excitation “Real” emission

Dapi 360 – 365 nm 445 – 475 nm

GFP 477 – 510 nm 510 – 540 nm

mCherry 551 – 599 nm 604 – 660 nm

CY5 590 – 650 nm 670 – 738 nm

Qdot (655 and 705) 440 - 482 nm 626 – 749 nm

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 Large image acquisition
• Before you start an automatic large image acquisition, make sure the
exposure time and LED intensity is correct for all the channels you want to
acquire (see page 13).
• Find “scan large image” under the “acquire” menu (this has more options
than through the ND acquisition mode. If you need multichannel capture,
define your channels (A).
• Define the size of the field (B) and placement of start position (C).
• Choose which objective you want to use (D).
• Define how you want to focus (E).

B
A
C

New autofocus with fluo

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 Timelapse acquisition
• Open the ND acquisition window (icon on the top menu).
• Check the “Time” flag (A) and define “intervals” at which the images should
be taken. Define the “duration” (B) of the experiment.
• For long term experiments, use PFA (perfect focus system ) or autofocus
(remember to start up the heating at least 0,5-1 hour before imaging, se
page 7).
• If you want to image multichannels or/and multipositions, remember to check
the flag and set up these too (D).
• You can define where the data is going to be saved (E), preferably Biomic.

D
A

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 Multi-point acquisition

• Open the ND acquisition window (icon on the top menu).

• Check the “multipoint” flag under ND acquisition (A).
• Define your multipoint positions in the XY window with a tick into an empty
square (arrow). Remember to check “include Z” (B) if your positions are
far apart.
• Define your channels (C) and interval (D) of your timelapse.
• For long term experiments, use PFA (perfect focus system ) or autofocus.
• You can define where the data is going to be saved (E), preferably Biomic.
• Remember to start the heating 0,5-1 hour before imaging (see page 7).

D A C

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 Z-acquisition

This is not frequently used as it requires an additional module in NIS-

Element to handle these images.

• Open the ND acquisition window (icon on the top menu).

• Check the “Z” flag under ND acquisition (A).
• Move through your sample in z and define the “top” and “bottom”.
• Define the step size (C) or leave it as default (according to Nyquist
sampling).
• You can define where the data is going to be saved (D), preferably Biomic.

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 Adding a scale bar

• The images you acquire on the system will have information about
the pixel size incorporated into the metadata due to the objective
calibration. You can therefore add a scale bar later and in any other
software.
• If you need a scale bar burned (it’s the only way) into your image,
click on the scale bar icon on the left tool bar menu.
• A new window will open where you can customize the design.

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 Where should I save my data

We prefer that you do not use USB keys and external hard drive on the
system. The pc is on the net, so there are a few options.

• Save onto Biomic. (\\klient.uib.no\felles\mofa\biomic). Ask MIC personnel

if you would like access.

• Save on to your cloud/one drive. Go to any browser and search for “office
365” and sign in using your email. In the left menu you will see the cloud
icon. Here you have access to 1 TB.

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